Spred-2 steady-state levels are regulated by phosphorylation and Cbl-mediated ubiquitination

Biochem Biophys Res Commun. 2006 Dec 29;351(4):1018-23. doi: 10.1016/j.bbrc.2006.10.150. Epub 2006 Nov 3.


Spred proteins modulate growth factor receptor signaling by inhibiting the Ras-MAPK cascade. Here, we show that Spred-1, Spred-2, and Spred-3 are ubiquitinated in HEK293T cells stimulated with epidermal growth factor (EGF) or pervanadate. Spred-2 tyrosines Y228 and/or Y231 in the Kit binding domain were identified as putative phosphorylation site(s) critical for Spred-2 ubiquitination. Depletion of Cbl and Cbl-b E3 ubiquitin ligases by RNA interference, or overexpression of a Cbl dominant inhibitory mutant (Cbl-N), inhibited Spred-2 ubiquitination, while conversely, wild type Cbl enhanced Spred-2 ubiquitination. Interaction of Spred-2 with Cbl-N was detectable by co-immunoprecipitation and required the Cbl SH2 domain and Spred-2 Y228 and Y231 residues. Studies on endogenous Spred-2 in ME4405 melanoma cells showed that pervanadate induced Spred-2 ubiquitination and a marked reduction in Spred-2 steady-state levels that was partially blocked by the proteasomal inhibitor, MG-132. These results suggest a role for Spred-2 tyrosine phosphorylation and ubiquitination in controlling Spred-2 expression levels.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adaptor Proteins, Signal Transducing
  • Cells, Cultured
  • Epidermal Growth Factor / pharmacology
  • Humans
  • Intracellular Signaling Peptides and Proteins / metabolism
  • Membrane Proteins
  • Phosphorylation
  • Protein Kinase Inhibitors / pharmacology
  • Protein Tyrosine Phosphatases / antagonists & inhibitors
  • Proto-Oncogene Proteins c-cbl / antagonists & inhibitors
  • Proto-Oncogene Proteins c-cbl / genetics
  • Proto-Oncogene Proteins c-cbl / metabolism*
  • RNA Interference
  • Repressor Proteins / metabolism*
  • Tyrosine / metabolism
  • Ubiquitin / metabolism*
  • Vanadates / pharmacology


  • Adaptor Proteins, Signal Transducing
  • Intracellular Signaling Peptides and Proteins
  • Membrane Proteins
  • Protein Kinase Inhibitors
  • Repressor Proteins
  • SPRED1 protein, human
  • SPRED2 protein, human
  • SPRED3 protein, human
  • Ubiquitin
  • pervanadate
  • Vanadates
  • Tyrosine
  • Epidermal Growth Factor
  • Proto-Oncogene Proteins c-cbl
  • Protein Tyrosine Phosphatases
  • CBL protein, human